CN107142228A - A kind of preparation of Escherichia coli outer membrane vesicles, medicine-carrying method and its application in antitumor - Google Patents
A kind of preparation of Escherichia coli outer membrane vesicles, medicine-carrying method and its application in antitumor Download PDFInfo
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- CN107142228A CN107142228A CN201710252957.9A CN201710252957A CN107142228A CN 107142228 A CN107142228 A CN 107142228A CN 201710252957 A CN201710252957 A CN 201710252957A CN 107142228 A CN107142228 A CN 107142228A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
Abstract
The invention discloses a kind of preparation of Escherichia coli outer membrane vesicles, medicine-carrying method and its application in antitumor.Preparing for the Escherichia coli outer membrane vesicles is as follows：In vitro culture uses LB culture mediums, cultivate e. coli strain bl21, after the culture supernatants without Escherichia coli are obtained after centrifugation, filtering, hyperfiltration treatment, it is final to centrifuge culture supernatants using ultracentrifuge, prepare Escherichia coli outer membrane vesicles, preparation-obtained Escherichia coli outer membrane vesicles uniform particle diameter, about 50nm or so.Under normal circumstances, bacterial outer membrane vesicles yield poorly, and carry medicine difficult.The present invention works out a kind of medicine-carrying method of brand-new directed toward bacteria outer membrane vesicles, the efficiency of carrying anti-tumor medicine can be significantly improved, so as to increase the inhibitory action to tumor cell proliferation and invasion and attack, bacterial outer membrane vesicles are shown as the good application prospect of new non-viral pharmaceutical carrier.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of preparation of Escherichia coli outer membrane vesicles, medicine-carrying method with
Its application in antitumor.
Background technology
Bacterial outer membrane vesicles (OMV) are the globular preteins liposomes of a kind of nano-scale, bilayer.Its particle diameter about exists
Between 50-100nm.Outer membrane vesicles outer surface is wrapped by phospholipid bilayer, rich in water-solubility protein, mRNA and microRNA etc.
Material.It is main outside Gram-negative bacteria secretion and outer discharge bacterium, is the important channel of bacterial metabolism process, simultaneously
It is also the important medium that signal transduction is carried out between bacterium.Gram-negative bacteria in the spontaneous release outer membrane vesicles of its growth period,
However, the bacterial outer membrane vesicles yield being spontaneously generated is relatively low, certain matter could be extracted by generally requiring to cultivate substantial amounts of bacterium
The outer membrane vesicles of amount.Using a series of detergent-treatment or ultrasonic processing method, can by destroy complete bacterium and then
Bacterium is promoted to produce OMV, but such method technique is more complicated, often also needs to multi-step purification, removes chemical reagent, or need
It is additional ultrasonically treated.
In terms of applied to pharmaceutical carrier, traditional pharmaceutical carrier particle diameter and toxicity are larger, synthesize relative complex, easily quilt
Macrophage etc. swallows, and carrier can not effectively reach tumor locus.Conventional medicament carrier includes cationic-liposome and cation
The major class of polymer two, their surfaces can be combined due to the presence of permanent positive charge by electrostatic interaction with medicine, but
Be cationic-liposome after intravenous injection, can be combined with negatively charged haemocyanin, cause targeting poor and can not
Circulate for a long time in vivo, and outer membrane vesicles are as a kind of pharmaceutical carrier of membrane structure, by its surface is negatively charged, particle diameter is small
And the features such as good biocompatibility, can for a long time be circulated in vivo, subsequent passive target is arrived by carrying anti-tumor medicine
Up to tumor locus, deliver the medicament in tumour cell.
The method delivered the medicament at present in OMV carriers has three kinds of 37 DEG C of incubation methods, ultrasonic method and electroporation.37℃
Incubation method by the cell intermembrane space and concentration gradient of itself make medicine from outside OMV to OMV diffusion insides, but OMV iuntercellulars
Gap very little and for fat-soluble film, so diffuser efficiency is very low, causes its drugloading rate low.Ultrasonic method and electroporation can be certain
OMV drugloading rate is improved in degree, but is needed by Ultrasound Instrument and electroporation apparatus, inconvenient operation, these limitations are hindered
Applications of the OMV in terms of pharmaceutical carrier.
The content of the invention
Not enough for more than, the present invention provides a kind of preparation of Escherichia coli outer membrane vesicles, medicine-carrying method and swollen with it anti-
Application in knurl, has directly used a kind of bacterial outer membrane vesicles derivant, can stimulate the more OMV of E. coli secretion,
Greatly improve Escherichia coli OMV yield.The present invention directly uses a kind of saponins small-molecule substance, n-dodecyl-β-D-maltoside
Glycosides, after it is incubated with OMV, can form poroid passage on OMV surfaces, small-molecule drug is entered by poroid channel permeability
Enter inside OMV, then take calcium chloride molecule to seal duct, be completely encapsulated in OMV.Method is simple and feasible, improves OMV
Carrier medicine carrying efficiency, and subsequent experimental proves that the OMV of carrying medicaments can substantially suppress tumour growth, has in pharmaceutical carrier field
Wide application prospect.
The technical proposal for solving the technical problem of the invention is as follows：A kind of preparation side of Escherichia coli outer membrane vesicles
Method, the preparation method is specific as follows：
(1) the Escherichia coli solution of in vitro culture is collected；
(2) Escherichia coli solution is centrifuged at 0-25 DEG C and discards precipitation, use membrane filtration supernatant, obtained without on bacterium
Clear liquid；
(3) obtained with super filter tube concentration step (2) without bacterial supernatant, the volume after concentration is the 1/50- before concentration
1/4；
(4) by after concentration without bacterial supernatant at 0-25 DEG C ultracentrifugation, supernatant discarding, precipitate with PBS buffer solutions
It is resuspended, repeats ultracentrifugation, abandoning supernatant, precipitation and be resuspended in PBS, finally produce bacterial outer membrane vesicles.
Further, the acquisition methods of Escherichia coli solution are as follows in the step (1)：
(1.1) frozen stock solution of BL21 bacterial strains is melted, using oese picking bacterial solution, be inoculated in containing 50 μ g/ml cards
1 single bacterium in the solid medium of that mycin, 37 DEG C of incubators on culture 1-48h, then picking solid medium is dropped down onto containing 50 μ
The LB fluid nutrient mediums of g/ml kanamycins, then the concussion and cultivate 1-24h at 37 DEG C；
(1.2) bacterial solution for then obtaining concussion and cultivate 1-24h is using volume ratio as 1：100 ratio is seeded to containing 50
In the LB fluid nutrient mediums of μ g/ml kanamycins, continue the concussion and cultivate at 37 DEG C, under 600nm wavelength, bacterium OD values
When reaching 0.1-0.5, isopropylthiogalactoside (IPTG) is added in LB fluid nutrient mediums, makes isopropylthio galactolipin
Ultimate density of the glycosides in LB fluid nutrient mediums is 1-500 μ g/ml, continues the concussion and cultivate 1-48h at 37 DEG C, final to obtain
Escherichia coli solution.
Further, the centrifugal rotational speed in the step (2) is 1000g-10000g, and centrifugation time is 1-30min.
Further, the aperture of the filter membrane is 0.2-1 μm.
Further, the ultracentrifugation rotating speed in the step (4) is 100000g-200000g, and centrifugation time is 0.5-
5h。
It is a further object of the present invention to provide a kind of medicine-carrying method of Escherichia coli outer membrane vesicles, this method utilizes dodecane
Antineoplastic is loaded into Escherichia coli outer membrane vesicles by base maltoside as perforating agent, obtains the thin of carrying anti-tumor medicine
Bacterium outer membrane vesicles.
Further, this method detailed process is as follows：
(a) Escherichia coli outer membrane vesicles are dispersed in PBS, then add Lauryl.beta.-maltoside thereto,
The mass ratio of Escherichia coli outer membrane vesicles, Lauryl.beta.-maltoside and PBS is 0.1-10： 0.1-10：1000,
0.1-10h is shaked at 37 DEG C；
(b) antineoplastic is added into solution made from step (a), the ultimate density for making antineoplastic is 1-
10mg/ml, 0.1-10h is incubated under the conditions of 37 DEG C；
(c) calcium chloride is added in the solution obtained to step (b), it is 0.1-1mg/ml to make calcium chloride concentration, is continued 37
0.1-10h is incubated under the conditions of DEG C, ultracentrifugation, abandoning supernatant, precipitation is resuspended in PBS, repeats ultracentrifugation, discard
Supernatant, precipitation are resuspended in PBS, that is, obtain the bacterial outer membrane vesicles of carrying anti-tumor medicine.
Further, the antineoplastic is doxorubicin hydrochloride, DNA, taxol, cis-platinum, mRNA, Gefitinib, Ji
One or more in western his shore, siRNA, mustargen, Pentostatin, Anastrozole and Rituximab are mixed by any ratio.
Further, the ultracentrifugation rotating speed in the step (c) is 100000g-200000g, and centrifugation time is 0.5-
5h。
The carrying anti-tumor medicine that the medicine-carrying method that the present invention also provides a kind of above-mentioned Escherichia coli outer membrane vesicles is obtained
Application of the bacterial outer membrane vesicles in antitumor.
Compared with prior art, the invention has the advantages that：
1) present invention improves Escherichia coli OMV yield using a kind of simple and feasible mode first, it is simple to operate, into
This relatively low, feasibility is high.
2) present invention according to the features such as OMV itself higher biocompatibilities, can carrying anti-tumor medicine, with it
Its conventional medicament carrier is compared, and toxicity can be reduced in theory, increases the pharmaceutical carrier interior circulation time, preferably by by moving-target
To mode reach tumor locus.
3) OMV can not only carry small molecule, anti-tumor drug, additionally it is possible to carrying gene class medicine, such as siRNA,
While growth of tumour cell is suppressed, the invasive ability of tumour cell can also be reduced, is a kind of multi-functional pharmaceutical carrier.
4) compared with the existing medicine-carrying methods of OMV, the present invention uses Lauryl.beta.-maltoside as promotion and carries medicine first
Reagent, and filter out it is rational be incubated concentration, compared with other methods, the present invention is simple to operate, and OMV film knot is not destroyed
Structure, is a kind of efficient medicine-carrying method.
Brief description of the drawings
Fig. 1 is the grain-size graph of outer membrane vesicles；
Fig. 2 is the scanning electron microscope (SEM) photograph of outer membrane vesicles；
Fig. 3 is the scanning electron microscope (SEM) photograph of outer membrane vesicles after amplification；
Fig. 4 is the Yield mapping that outer membrane vesicles are made in different modes；
Fig. 5 is the different load medicine design sketch for carrying prescription formula of three kinds of outer membrane vesicles；
Fig. 6 is the intake design sketch of the external membrane vesicle of tumour cell；
Fig. 7 is inhibition figure of the load medicine outer membrane vesicles to tumour cell pair；
Fig. 8 is inhibition figure of the load medicine outer membrane vesicles to the invasive ability of tumour cell pair.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described further.
Embodiment 1：
The preparation of Escherichia coli outer membrane vesicles
(1) recovery of Escherichia coli and in vitro culture
The frozen stock solutions of BL21 bacterial strains is melted, using oese picking bacterial solution, is inoculated in containing 50 μ g/ml cards that is mould
1 single bacterium drops down onto containing 50 μ g/ml cards that is mould on culture 1h in the solid medium of element, 37 DEG C of incubators, inoculation solid medium
The LB fluid nutrient mediums of element, 37 DEG C of concussion and cultivates 1 hour.
Then by bacterial solution using volume ratio as 1：100 ratio is seeded to the LB liquid training containing 50 μ g/ml kanamycins
Base (by high-temperature sterilization) is supported, continues 37 DEG C of concussion and cultivates, when bacterium OD values (600nm) reach 0.1, in the training of LB liquid
Support and isopropylthiogalactoside (IPTG) is added in base, make isopropylthiogalactoside final in LB fluid nutrient mediums
Concentration is 1 μ g/ml, promotes E. coli secretion outer membrane vesicles, continues the concussion and cultivate 1h at 37 DEG C.
(2) Separation ＆ Purification of Escherichia coli outer membrane vesicles
The culture of Escherichia coli of in vitro culture is collected, at 0 DEG C, 1000g centrifugation 1min discard precipitation, with 0.2 μm of filter
Membrane filtration supernatant, is obtained without bacterial supernatant.Using super filter tube be concentrated by ultrafiltration supernatant, after being concentrated without bacterial supernatant
Liquid, the volume after concentration is 1/50 before concentration.At 0 DEG C, 100000g ultracentrifugation 0.5h, supernatant discarding is precipitated with PBS
Buffer solution is resuspended, again with 100000g ultracentrifugation 0.5h, abandoning supernatant, and precipitation is resuspended in PBS, that is, obtains thin
Bacterium outer membrane vesicles.
The load medicine of Escherichia coli outer membrane vesicles
Obtained Escherichia coli outer membrane vesicles are dispersed in PBS, n-dodecyl-β-D-maltoside is added thereto
Glycosides, the mass ratio of Escherichia coli outer membrane vesicles, Lauryl.beta.-maltoside and PBS is 0.1： 0.1：1000, at 37 DEG C
Under shake 0.1h.Antineoplastic is added in vesicle solution to outside Escherichia coli, the ultimate density for making antineoplastic is 1mg/
Ml, is incubated 0.1h under the conditions of 37 DEG C.Antineoplastic can gradually spread from high concentration gradient to low concentration gradient, into large intestine bar
In bacterium outer membrane vesicles.
In order to prevent medicine from revealing, it is necessary to add chlorination calcium ion into solution, it is 0.1 mg/ml to make calcium chloride concentration,
0.1h is incubated under the conditions of continuing 37 DEG C, ultracentrifugation, abandoning supernatant, precipitation is resuspended in PBS buffer solutions.Repeat ultracentrifugation,
Abandoning supernatant, precipitation is resuspended in PBS, that is, obtains the bacterial outer membrane vesicles of carrying anti-tumor medicine.
Embodiment 2：
The preparation of Escherichia coli outer membrane vesicles
(1) recovery of Escherichia coli and in vitro culture
The frozen stock solutions of BL21 bacterial strains is melted, using oese picking bacterial solution, is inoculated in containing 50 μ g/ml cards that is mould
1 single bacterium drops down onto containing 50 μ g/ml cards that is mould on culture 48h in the solid medium of element, 37 DEG C of incubators, inoculation solid medium
The LB fluid nutrient mediums of element, 37 DEG C of concussion and cultivates 24 hours.
Then by bacterial solution using volume ratio as 1：100 ratio is seeded to the LB liquid training containing 50 μ g/ml kanamycins
Base (by high-temperature sterilization) is supported, continues 37 DEG C of concussion and cultivates, when bacterium OD values (600nm) reach 0.5, in the training of LB liquid
Support and isopropylthiogalactoside (IPTG) is added in base, make isopropylthiogalactoside final in LB fluid nutrient mediums
Concentration is 500 μ g/ml, promotes E. coli secretion outer membrane vesicles, continues concussion and cultivate 48h.
(2) Separation ＆ Purification of Escherichia coli outer membrane vesicles
The culture of Escherichia coli of in vitro culture is collected, at 25 DEG C, 10000g centrifugation 30min discard precipitation, 1 μm of filter
Membrane filtration supernatant, is obtained without bacterial supernatant.Using super filter tube be concentrated by ultrafiltration supernatant, after being concentrated without bacterial supernatant
Liquid, the volume after concentration is 1/4 before concentration.At 25 DEG C, 200000g ultracentrifugation 5h, supernatant discarding, precipitation is slow with PBS
Fliud flushing is resuspended, again ultracentrifugation, abandoning supernatant, and precipitation is resuspended in PBS, that is, the bacterial outer membrane capsule prepared
Bubble.
The load medicine of Escherichia coli outer membrane vesicles
Obtained Escherichia coli outer membrane vesicles are dispersed in PBS, n-dodecyl-β-D-maltoside is added thereto
Glycosides, the mass ratio of Escherichia coli outer membrane vesicles, Lauryl.beta.-maltoside and PBS is 10： 10：1000, at 37 DEG C
Shake 10h.Antineoplastic is added in vesicle solution to outside Escherichia coli, the ultimate density for making antineoplastic is 10mg/ml,
10h is incubated under the conditions of 37 DEG C.Antineoplastic can gradually spread from high concentration gradient to low concentration gradient, into outside Escherichia coli
In membrane vesicle.
In order to prevent medicine from revealing, it is necessary to add chlorination calcium ion into solution, it is 1mg/ml to make calcium chloride concentration, is continued
10h is incubated under the conditions of 37 DEG C, ultracentrifugation, abandoning supernatant, precipitation is resuspended in PBS.Repeat 200000g ultracentrifugations
5h, abandoning supernatant, precipitation is resuspended in PBS, that is, obtains the bacterial outer membrane vesicles of carrying anti-tumor medicine.
The antineoplastic used in embodiment 1 and embodiment 2 is doxorubicin hydrochloride, DNA, taxol, cis-platinum,
One or more in mRNA, Gefitinib, gemcitabine, siRNA, mustargen, Pentostatin, Anastrozole and Rituximab
It is mixed by any ratio.
Embodiment 3：
The checking of Escherichia coli outer membrane vesicles
(1) grain diameter measurement is carried out to Escherichia coli outer membrane vesicles
The synthetic outer membrane vesicles of embodiment 1 are adjusted to 100 μ g/ml, in Malvern laser first by PBS
The size of outer membrane vesicles is measured under particle instrument, as a result as shown in Figure 1.
(2) externally membrane vesicle carries out morphologic observation
The outer membrane vesicles solution of Example 1, is added dropwise, directly under ESEM observation outer membrane vesicles online in special purpose copper
Size and form, as a result as shown in Figure 2 and Figure 3.
(3) Escherichia coli outer membrane vesicles yield is investigated
The outer membrane vesicles solution of Example 1, adds lysozyme (1mg/ml), room temperature shakes 30min.Then use BCA eggs
White kit detection protein concentration, that is, obtain outer membrane vesicles concentration obtained by embodiment 1.
Outer membrane vesicles solution obtained by the Escherichia coli without isopropylthiogalactoside (IPTG) processing is taken, is added molten
Bacterium enzyme (1mg/ml), room temperature shakes 30min.Then using BCA protein reagents box detection protein concentration, that is, obtain without isopropyl
The outer membrane vesicles concentration of thiogalactoside (IPTG) processing.The yield of outer membrane vesicles prepared by two kinds of different cultural methods
As shown in Figure 4.
(4) carrier medicine carrying efficiency of distinct methods Escherichia coli outer membrane vesicles is investigated
Outer membrane vesicles solution containing medicine made from Example 1, adds lysozyme (1mg/ml), room temperature is shaked
30min.The concentration of drug in solution is then detected using high performance liquid chromatography, and compared with total dosage, draws chemistry
The carrier medicine carrying efficiency of medicine.
Outer membrane vesicles solution made from Example 1, adds medicine (1mg/ml), and 10min is incubated with outer membrane vesicles solution,
Then using electroporation apparatus (Eppendorf AG, Hamburg, Germany), with 1000kV electric shock 5ms, finally in 37 DEG C of bars
30min is incubated under part, the outer membrane vesicles solution for carrying medicine is produced.Above-mentioned outer coating solution is taken, lysozyme (1mg/ml), room temperature shake is added
Shake 30min.The concentration of drug in solution is then detected using high performance liquid chromatography, and compared with total dosage, draws electricity
The drug delivery efficiency that perforation method is prepared.
Outer membrane vesicles solution made from Example 1, adds medicine (1mg/ml), is incubated 24h under the conditions of 37 DEG C, produces
Carry the outer membrane vesicles solution of medicine.Above-mentioned outer coating solution is taken, lysozyme (1mg/ml) is added, room temperature shakes 30min.Then using high
Effect liquid phase chromatogram method detects the concentration of drug in solution, and compared with total dosage, draws what 37 DEG C of incubation methods were prepared
Drug delivery efficiency.
The carrier medicine carrying efficiency that three kinds of different medicine-carrying methods prepare outer membrane vesicles is as shown in Figure 5.
(5) the cellular uptake effect for the outer membrane vesicles solution for carrying medicine is investigated
With every hole 2 × 104The B16F10 cell kinds of logarithmic growth in Tissue Culture Plate, are placed in thin by the density of individual cell
After born of the same parents' incubator culture 24 hours, remove nutrient solution and washed twice with PBS, the high sugar of DMEMs of the 500 μ l without serum is added per hole
Nutrient solution.The outer membrane vesicles solution for carrying medicine is diluted with 5wt% aqueous sucrose solutions, said medicine solution is added, makes its cell drug
Incubation be 10 μ g/ml.In 37 DEG C, 5wt%CO2Under the conditions of with after B16F10 cell incubations 12 hours, remove nutrient solution, add
The paraformaldehydes of 200 μ l 4% fix 30min, and PBS is washed 2 times, and each 10min, each sample adds 200 μ l DAPI (1 μ g/ml)
Dyeing liquor, room temperature lucifuge reaction 10min, PBS is washed 2 times, aobvious using fluorescence immediately under conditions of excitation wavelength is 550nm
Micro mirror observes the distribution situation of fluorescence.As a result it is as shown in Figure 6.
(6) antitumous effect for the outer membrane vesicles solution for carrying medicine is investigated
With every hole 1 × 104The B16F10 cells kind of logarithmic growth in 96 orifice plates, is placed in cell training by the density of individual cell
After supporting case culture 24 hours, remove nutrient solution and washed twice with PBS, the high sugar cultures of DMEMs of the 100 μ l without serum are added per hole
Liquid.The outer membrane vesicles solution for carrying medicine is diluted with 5wt% aqueous sucrose solutions, said medicine solution is added, makes incubating for its cell drug
It is respectively 1 μ g/ml, 5 μ g/ml and 10 μ g/ml to educate concentration.In 37 DEG C, 5wt%CO2Under the conditions of with B16F10 cell incubations 24
After hour, it is 5mg/ml MTT solution that 20 μ l concentration are added per hole, and nutrient solution is removed after being incubated 4h, and 150 μ l are added per hole
DMSO, shaking makes after crystallization is completely dissolved, in determining each group OD values under 570nm on ELIASA.As a result as shown in Fig. 7.
(7) influence of the outer membrane vesicles solution for carrying medicine to cell invasion is investigated
B16F10 vitro invasion ability is investigated with Transwell culture plates (diameter 6.5mm, 8 μm of aperture).By B16F10
Cell, according to every hole 5 × 104The density kind of individual cell is in 24 orifice plates, after being placed in cell culture incubator culture 24 hours, discards training
Nutrient solution is simultaneously washed twice with PBS, and the high sugared nutrient solutions of DMEMs of the 500 μ l without serum are added per hole.Diluted with 5wt% aqueous sucrose solutions
The outer membrane vesicles solution of medicine is carried, its drug concentration is adjusted to 100 μ g/ml.Add the 40 above-mentioned solution of μ l, 5wt%CO2Under the conditions of after
Continuous culture 24 hours.Then by cell with every hole 3 × 104Concentration add Transwell plates upper strata, lower floor add 600 μ
L contains the nutrient solution of 10wt% serum, continues to cultivate 48h, takes out upper strata plug-in unit, the cell on film upper strata is gently wiped with cotton swab,
The cell for attacking lower floor fixes 20min with 4wt% paraformaldehydes, and PBS is washed three times, cell violet staining.Use fluorescence
The invasion and attack situation of micro- sem observation cell, as a result as shown in Figure 8.
Claims (10)
1. a kind of preparation method of Escherichia coli outer membrane vesicles, it is characterised in that the preparation method is specific as follows：
(1) the Escherichia coli solution of in vitro culture is collected.
(2) Escherichia coli solution is centrifuged at 0-25 DEG C and discards precipitation, use membrane filtration supernatant, obtained without bacterial supernatant
Liquid.
(3) obtained with super filter tube concentration step (2) without bacterial supernatant, the volume after concentration is the 1/50-1/4 before concentration.
(4) by after concentration without bacterial supernatant at 0-25 DEG C ultracentrifugation, supernatant discarding, precipitation be resuspended with PBS,
Repeat ultracentrifugation, abandoning supernatant, precipitation and be resuspended in PBS, finally produce bacterial outer membrane vesicles.
2. the preparation method of the outer vesica of escherichia coli membrane according to claim 1, it is characterised in that big in the step (1)
The acquisition methods of enterobacteria solution are as follows：
(1.1) frozen stock solutions of BL21 bacterial strains is melted, using oese picking bacterial solution, is inoculated in containing 50 μ g/ml cards that is mould
1 single bacterium in the solid medium of element, 37 DEG C of incubators on culture 1-48h, then picking solid medium is dropped down onto containing 50 μ g/ml
The LB fluid nutrient mediums of kanamycins, then the concussion and cultivate 1-24h at 37 DEG C；
(1.2) bacterial solution for then obtaining concussion and cultivate 1-24h is using volume ratio as 1：100 ratio is seeded to containing 50 μ g/
In the LB fluid nutrient mediums of ml kanamycins, continue the concussion and cultivate at 37 DEG C, under 600nm wavelength, bacterium OD values reach
During 0.1-0.5, isopropylthiogalactoside (IPTG) is added in LB fluid nutrient mediums, isopropylthiogalactoside is existed
Ultimate density in LB fluid nutrient mediums is 1-500 μ g/ml, continues the concussion and cultivate 1-48h at 37 DEG C, final to obtain large intestine bar
Bacterium solution.
3. the preparation method of Escherichia coli outer membrane vesicles according to claim 1, it is characterised in that in the step (2)
Centrifugal rotational speed be 1000g-10000g, centrifugation time is 1-30min.
4. the preparation method of Escherichia coli outer membrane vesicles according to claim 1, it is characterised in that the aperture of the filter membrane
For 0.2-1 μm.
5. the preparation method of Escherichia coli outer membrane vesicles according to claim 1, it is characterised in that in the step (4)
Ultracentrifugation rotating speed be 100000g-200000g, centrifugation time is 0.5-5h.
6. a kind of medicine-carrying method of Escherichia coli outer membrane vesicles, it is characterised in that this method is made using Lauryl.beta.-maltoside
Antineoplastic is loaded into the preparation-obtained Escherichia coli outer membrane vesicles of claim 1 for perforating agent, obtains carrying anti-swell
The bacterial outer membrane vesicles of tumor medicine.
7. the medicine-carrying method of Escherichia coli outer membrane vesicles according to claim 6, it is characterised in that this method detailed process
It is as follows：
(a) Escherichia coli outer membrane vesicles are dispersed in PBS, then add Lauryl.beta.-maltoside, large intestine thereto
The mass ratio of bacillus outer membrane vesicles, Lauryl.beta.-maltoside and PBS is 0.1-10：0.1-10：1000, at 37 DEG C
Shake 0.1-10h；
(b) antineoplastic is added into solution made from step (a), the ultimate density for making antineoplastic is 1-10mg/ml,
0.1-10h is incubated under the conditions of 37 DEG C；
(c) calcium chloride is added in the solution obtained to step (b), it is 0.1-1mg/ml to make calcium chloride concentration, is continued in 37 DEG C of bars
0.1-10h is incubated under part, ultracentrifugation, abandoning supernatant, precipitation is resuspended in PBS, repeats ultracentrifugation, supernatant discarding
Liquid, precipitation are resuspended in PBS, that is, obtain the bacterial outer membrane vesicles of carrying anti-tumor medicine.
8. the medicine-carrying method of the Escherichia coli outer membrane vesicles according to claim 6 or 7, it is characterised in that described antitumor
Medicine be doxorubicin hydrochloride, DNA, taxol, cis-platinum, mRNA, Gefitinib, gemcitabine, siRNA, mustargen, Pentostatin,
One or more in Anastrozole and Rituximab are mixed by any ratio.
9. the medicine-carrying method of Escherichia coli outer membrane vesicles according to claim 7, it is characterised in that in the step (c)
Ultracentrifugation rotating speed be 100000g-200000g, centrifugation time is 0.5-5h.
What 10. the medicine-carrying method of the Escherichia coli outer membrane vesicles according to claim any one of 6-9 was obtained carries antitumor
Application of the bacterial outer membrane vesicles of medicine in antitumor.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108014093A (en) * | 2017-12-15 | 2018-05-11 | 东南大学 | It is a kind of to be used to treat nanometer formulation of inflammatory bowel disease and its preparation method and application |
| CN108587998A (en) * | 2018-05-17 | 2018-09-28 | 浙江大学 | A kind of excretion body, the preparation method of excretion body and its application in the drug for preparing skin superficial tumour |
| CN110037996A (en) * | 2019-04-30 | 2019-07-23 | 郑州大学 | Endogenous height expresses the preparation method and its application in preparation of anti-tumor drugs of membrane vesicle in the Escherichia coli of miRNA |
| CN110079473A (en) * | 2019-03-28 | 2019-08-02 | 北京市农林科学院 | A kind of preparation method and applications of pair chicken poultry bacillus outer membrane vesicles |
| CN110804621A (en) * | 2019-10-31 | 2020-02-18 | 郑州大学 | Preparation method of escherichia coli extracellular vesicle with endogenous high-expression miRNA (micro ribonucleic acid) |
| CN111454979A (en) * | 2020-04-03 | 2020-07-28 | 江苏省农业科学院 | Method for improving yield of bacterial outer membrane vesicles and application thereof |
| CN112410212A (en) * | 2019-08-22 | 2021-02-26 | 四川大学 | Production system, separation and purification system and method of bacterial membrane vesicles |
| CN112891318A (en) * | 2019-12-03 | 2021-06-04 | 复旦大学 | Adriamycin nano-particle encapsulated by bacterial outer membrane vesicle and application thereof |
| CN113230414A (en) * | 2021-06-10 | 2021-08-10 | 曲阜师范大学 | Biological nano drug delivery system for accurately targeting lung tumor cells and preparation method and application thereof |
| CN113388520A (en) * | 2021-06-17 | 2021-09-14 | 中国科学院城市环境研究所 | Purification method of extracellular vesicles |
| CN114288270A (en) * | 2022-01-20 | 2022-04-08 | 中山大学·深圳 | Calcium carbonate-polydopamine coated drug-loaded bacteria outer membrane vesicle nanoparticle and preparation method and application thereof |
| CN114668742A (en) * | 2022-03-17 | 2022-06-28 | 中国科学技术大学 | Bacteria-like nano-drug delivery system and preparation method and application thereof |
| CN116268419A (en) * | 2023-03-13 | 2023-06-23 | 大连工业大学 | Probiotic nano-vesicles, preparation method thereof and application thereof in embedding bioactive substances |
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Cited By (20)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108014093B (en) * | 2017-12-15 | 2020-09-11 | 东南大学 | Nanometer medicinal preparation for treating inflammatory bowel disease, and its preparation method and application |
| CN108014093A (en) * | 2017-12-15 | 2018-05-11 | 东南大学 | It is a kind of to be used to treat nanometer formulation of inflammatory bowel disease and its preparation method and application |
| CN108587998A (en) * | 2018-05-17 | 2018-09-28 | 浙江大学 | A kind of excretion body, the preparation method of excretion body and its application in the drug for preparing skin superficial tumour |
| CN108587998B (en) * | 2018-05-17 | 2021-05-18 | 浙江大学 | Exosome, preparation method of exosome and application of exosome in preparation of medicine for treating skin superficial tumors |
| CN110079473A (en) * | 2019-03-28 | 2019-08-02 | 北京市农林科学院 | A kind of preparation method and applications of pair chicken poultry bacillus outer membrane vesicles |
| CN110037996A (en) * | 2019-04-30 | 2019-07-23 | 郑州大学 | Endogenous height expresses the preparation method and its application in preparation of anti-tumor drugs of membrane vesicle in the Escherichia coli of miRNA |
| CN110037996B (en) * | 2019-04-30 | 2022-01-18 | 郑州大学 | Preparation method of escherichia coli inner membrane vesicle of endogenous high-expression miRNA and application of escherichia coli inner membrane vesicle in preparation of antitumor drugs |
| CN112410212A (en) * | 2019-08-22 | 2021-02-26 | 四川大学 | Production system, separation and purification system and method of bacterial membrane vesicles |
| CN112410212B (en) * | 2019-08-22 | 2022-03-01 | 四川大学 | Production system, separation and purification system and method of bacterial membrane vesicles |
| CN110804621A (en) * | 2019-10-31 | 2020-02-18 | 郑州大学 | Preparation method of escherichia coli extracellular vesicle with endogenous high-expression miRNA (micro ribonucleic acid) |
| CN112891318A (en) * | 2019-12-03 | 2021-06-04 | 复旦大学 | Adriamycin nano-particle encapsulated by bacterial outer membrane vesicle and application thereof |
| CN111454979A (en) * | 2020-04-03 | 2020-07-28 | 江苏省农业科学院 | Method for improving yield of bacterial outer membrane vesicles and application thereof |
| CN111454979B (en) * | 2020-04-03 | 2023-06-06 | 江苏省农业科学院 | Method for improving bacterial outer membrane vesicle yield and application thereof |
| CN113230414A (en) * | 2021-06-10 | 2021-08-10 | 曲阜师范大学 | Biological nano drug delivery system for accurately targeting lung tumor cells and preparation method and application thereof |
| CN113388520B (en) * | 2021-06-17 | 2023-03-14 | 中国科学院城市环境研究所 | Purification method of extracellular vesicles |
| CN113388520A (en) * | 2021-06-17 | 2021-09-14 | 中国科学院城市环境研究所 | Purification method of extracellular vesicles |
| CN114288270A (en) * | 2022-01-20 | 2022-04-08 | 中山大学·深圳 | Calcium carbonate-polydopamine coated drug-loaded bacteria outer membrane vesicle nanoparticle and preparation method and application thereof |
| CN114288270B (en) * | 2022-01-20 | 2023-04-14 | 中山大学·深圳 | Calcium carbonate-polydopamine coated drug-loaded bacteria outer membrane vesicle nanoparticle and preparation method and application thereof |
| CN114668742A (en) * | 2022-03-17 | 2022-06-28 | 中国科学技术大学 | Bacteria-like nano-drug delivery system and preparation method and application thereof |
| CN116268419A (en) * | 2023-03-13 | 2023-06-23 | 大连工业大学 | Probiotic nano-vesicles, preparation method thereof and application thereof in embedding bioactive substances |
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